The present invention relates to isolated polynucleotide molecules useful for analyzing alloantigen phenotypes, to peptides encoded by these molecules, and to the diagnostic and therapeutic uses thereof relating to a human platelet Bak polymorphism, including a method for typing platelet membrane glycoproteins which entails an analysis of amplified cDNA produced from platelet mRNA or of genomic DNA.
Blood obtained from different individuals has been found to have different antigenic and immune properties, to the extent that antibodies in the blood of one person may react with antigens on red blood cells or platelets in the blood of another individual. These antigens are often found on membrane glycoproteins present on the surface of the cells. These membrane glycoprotein antigens can induce the production of antibodies against them when they are introduced as foreign proteins in transfused blood or in fetal blood. Human platelets and red blood cells contain dozens of identifiable membrane glycoprotein constituents, only some of which have been well characterized.
Membrane glycoproteins which induce antibody production in same species are the called "alloantigens." Alloantigens have been characterized for both red blood cells and platelets. Recognized classes of red blood cell and platelet alloantigens have been described, over the past 30 years, based on observations of antibody reactions occurring when patients have been exposed to blood from other individuals. The lack of sequenceable antigen protein and clonable antigen-encoding mRNA has prevented molecular characterization of the different alleles coding for many clinically important alloantigens.
One system of alloantigens, consisting of the platelet Bak.sup.a and Bak.sup.b alloantigens, are carried by the human platelet membrane glycoprotein IIb-IIIa (GPIIb-GPIIIa) complex, which mediates platelet aggregation by providing functional receptors for fibrinogen on platelet surfaces. See Phillips, et al., Blood 71: 831-43 (1988). GPIIb and GPIIIa are known to bear a number of clinically important, alloantigenic determinants which are responsible for eliciting an immune response in two well-described clinical syndromes, post-transfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NATP). See Kunicki & Newman in CURRENT STUDIES IN HEMATOLOGY AND BLOOD TRANSFUSION 18-32 (1986); Aster in ADVANCES IN IMMUNOLOGY AND BONE MARROW TRANSPLANTATION 103-118 (1984).
The Bak alloantigen system is the second or third most frequently implicated stimulus in these disorders. There are two serologically defined, but molecularly undefined, allelic forms of the Bak alloantigen, designated "Bak.sup.a " and "Bak.sup.b," which are thought to be expression products of the GPIIb gene. von dem Borne, et al., Vox Sang. 39:113 (1980); Kickler, et al., Blood: 71(4):894 (1988); Keifel, et al., Vox Sang. 56:93 (1989). The gene frequencies for these two alleles have been calculated to be 61% for Bak.sup.a and 39% for Bak.sup.b, while the observed phenotypic frequencies are 37% for Bak.sup.a homozygous, 15% for Bak.sup.b homozygous, and 48% for heterozygous individuals. see Kickler, et al., Vox Sang. 56:93 (1989). Based upon these frequencies, the probability of fetal-maternal Bak incompatibility would be significant, but fewer than 5% of the cases of NATP (or 1/40,000) are attributable to Bak. This suggests that other factors contribute to the likelihood of developing NATP.
Determination of the amino acid sequence variations that are presumably responsible for forming the relevant epitopes of red blood cell and platelet alloantigens has been achieved in only a few instances, due largely to the formidable difficulties in obtaining protein-sequence information from those often large glycoproteins. In particular, the amino acid-sequence variation responsible for the relevant epitopes has not yet been reported for either the Bak.sup.a or Bak.sup.b forms of the 125 kilodalton (kd) GPIIb molecule.